The present invention relates to a glycosaminoglycan having sulfate groups, in which substantially all the sulfate groups bound to the 6-positions of the glucosamine residues constituting the glycosaminoglycan are removed and the removal of other sulfate groups is minimized, and to pharmaceuticals comprising the glycosaminoglycan as an active ingredient, in addition to a method for producing the glycosaminoglycan.
Heparin is one of glycosaminoglycans having as a backbone structure being composed of a repetitive structure of a disaccharide unit composed of a uronic acid (iduronic acid (IdoA) or glucuronic acid (GlcA)) residue and a glucosamine (GlcN) residue. Heparin is one of glycosaminoglycans in which the hydroxyl group at the 2-position of the uronic acid residue and the hydroxyl group at the 6-position and the amino group at the 2-position of the glucosamine residue each undergo a certain degree of sulfation. Because heparin has an antithorombin III (hereinafter also referred to as xe2x80x9cATIIIxe2x80x9d) binding site (FEBS Lett. (1980) 117, 203-206), and binds with ATIII to inhibit the action of the thorombin, thereby giving rise to anticoagulative action, heparin has long been extensively used as a pharmaceutical agent such as anticoagulants for improving the results of dialysis treatment and the like. More recently, it has been found that heparin interacts with various physiological active factors. For example, heparin interacts with lipoprotein lipase (J. Biol. Chem. (1981) 256, 12893-12898) and has an affinity for basic fibroblast growth factor (J. Cell Biol. (1990) 111, 1651-1659).
Under such a situation, attention has been focusing on the domain structures within heparin that take part in the binding between heparin and specific cell growth factors or cytokines. Moreover, considerable research is being conducted relating to chemical modifications represented by desulfation of heparin aiming at reducing the anticoagulative action due to the presence of the ATIII binding site to thereby increase the interaction with physiological active factors (J. Carbohydr. Chem. (1993) 12, 507-521; Carbohydr. Res. (1989) 193, 165-172; Carbohydr. Res. (1976) 46, 87-95; WO 95/30424, etc.).
With respect to the aforementioned desulfation of heparin, in recent years the focus has been on removing the sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue in the heparin (6-desulfation). As desulfation methods, there can be mentioned the method using solvolysis (WO 95/30424) and the method using a silylating reagent (WO 96/01278).
With the former method, along with the removal of the sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue in the heparin molecule (6-O-sulfate group), the sulfate group bound to the hydroxyl group at the 2-position of the uronic acid residue (2-O-sulfate group) and the sulfate group bound to the amino group at the 2-position of the glucosamine residue (N-sulfate group) are also removed. Thus, in the course of using the former method to remove substantially all of the 6-O-sulfate groups, almost all of the N-sulfate groups of the glucosamine residues and 2-O-sulfate groups of the uronic acid residues are also lost. While the amino group at the 2-position of the glucosamine residue of the heparin thus modified can be resulfated, resulfation of the 2-position of the uronic acid residue without sulfating the 6-position of the glucosamine residue is difficult.
The latter method is superior to the former method in that it enables a more specific removal of the 6-O-sulfate group of the glucosamine residue. However, with the modified heparin thus obtained with the latter method, the effective disaccharide yield as determined by the enzymatic disaccharide analysis method is low, which means that there is still a problem with the method in that the structural identification therefor may not be enough for pharmaceutical applications. Moreover, while the anticoagulative action of the modified heparin is greatly reduced, it is not absent, and it has not been possible to obtain modified heparin in which the anticoagulative activity has been completely eliminated.
Because heparin or fragments thereof have affinities for various physiological active substances and the affinities are closely related to the functions of such substances, intensive studies have been made for search and development of drugs utilizing heparin or modified heparin. However, despite such efforts, they have yet been used effectively only as a blood anticoagulation agent in pharmaceutical applications.
That is, in focusing on using heparin for applications other than as an anticoagulant, it is important to substantially eliminate its anticoagulation and hemorrhagic actions and, with respect to using it as a pharmaceutical substance, it has to enable its xe2x80x9cstructural identification as a substancexe2x80x9d. With respect to these problems, there must be further improvements, and it has been desired to resolve these remaining problems and utilize heparin""s affinities for physiologically active substances to provide drugs that are safe and useful.
As a result of assiduous studies aiming at resolution of the above problems, the present inventors succeeded in, by using a specific method to effect desulfation of glycosaminoglycans such as heparin that have sulfate groups, preparation of a novel glycosaminoglycan in which its anticoagulative and hemorrhagic activities were substantially eliminated while its biologically advantageous effects for living bodies such as its affinities for physiologically active substances were maintained, and unidentifiable structures were markedly reduced to the extent that the xe2x80x9cstructural identification of the substancexe2x80x9d can be readily attained, which is important in terms of pharmaceutical applications. Thus, the present invention has been accomplished.
Specifically, it was confirmed that a glycosaminoglycan obtained by subjecting a glycosaminoglycan having sulfate groups to heat treatment at 100xc2x0 C. or higher in pyridine in the presence of a silylating agent, N-methyl-N-(trimethylsilyl)-trifluoroacetamide (hereinafter also referred to as xe2x80x9cMTSTFAxe2x80x9d) to remove substantially all of the 6-O-sulfate groups from the glucosamine residues, then evaporating the pyridine from the reaction mixture, adding water and concentrating under reduced pressure, was highly effective in promoting the healing of skin wounds and treating diabetic skin ulcers, and had fructose-1,6-bis-phosphate aldolase inhibitory activity, and that its anticoagulative and hemorrhagic activities had disappeared.
Further, it was confirmed to be possible to readily specify the structure of the glycosaminoglycan prepared by the above method with precision by using an enzymatic disaccharide analysis method utilizing glycosaminoglycan-degrading enzymes, because of the good digestibility of the glycosaminoglycan with glycosaminoglycan-degrading enzymes. This is in contrast to the previous difficulty in identifying structures of modified heparins that had been modified by being subjected to various types of chemical treatment. Using the glycosaminoglycan of which structure can thus be identified as pharmaceuticals makes it possible to provide pharmaceuticals that are highly safe and useful.
The present inventors further found that the glycosaminoglycan had high affinity for fructose-1,6-bis-phosphate aldolase, a key enzyme in the glycolytic pathway, and could be used as a strong inhibitor of that enzyme. Thus, it has become possible to provide a novel fructose-1,6-bis-phosphate aldolase inhibitor.
That is, the present invention provides the followings.
1. A glycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groups, wherein substantially no sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue in the backbone structure is detected as determined by a chemical disaccharide analysis method in which the glycosaminoglycan is decomposed with nitrous acid, reacted with para-nitrophenylhydrazine and analyzed by high performance liquid chromatography, and the molar % of a uronic acid residue having a sulfate group at the 2-position is not less than 45%, relative to total uronic acid residues, the molar % being calculated from a disaccharide composition obtained by an enzymatic disaccharide analysis method in which the glycosaminoglycan is digested with glycosaminoglycan-degrading enzymes and analyzed by high performance liquid chromatography.
2. A glycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groups, wherein, in a disaccharide composition of the glycosaminoglycan obtained by an enzymatic disaccharide analysis in which the glycosaminoglycan is digested with glycosaminoglycan-degrading enzymes and analyzed by high performance liquid chromatography, the total of 2-acetamido-2-deoxy-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose, 2-deoxy-2-sulfamino-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose and 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose is not more than 10 mol %, and 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose is not more than 1.5 mol %, and an effective disaccharide yield is not less than 60%.
3. The glycosaminoglycan according to the item 2, wherein, in the disaccharide composition obtained by the enzymatic disaccharide analysis method, the total of 2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose, 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose and 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose is not less than 45 mol %.
4. The glycosaminoglycan according to any one of the items 1 to 3, wherein, in 13C-nuclear magnetic resonance spectrometry analysis of the glycosaminoglycan using a 5% solution of the glycosaminoglycan in deuterium oxide and sodium 3-(trimethylsilyl)propionate as a standard, substantially no peak is detected at 66.5 to 67.5 ppm and signal intensities around 100 ppm and 102 ppm are higher than signal intensity around 98.3 ppm.
5. A fructose-1,6-bis-phosphate aldolase inhibitor which comprises the glycosaminoglycan as defined in any one of the items 1 to 4 (hereinafter also referred to as xe2x80x9cthe glycosaminoglycan of the present inventionxe2x80x9d) as an active ingredient.
6. A pharmaceutical composition comprising the glycosaminoglycan of the present invention as an active ingredient.
7. The pharmaceutical composition according to the item 6, which is an agent for treatment of tissue wounds and ulcers.
8. The pharmaceutical composition according to the item 6, which is an agent for treating skin diseases.
9. The pharmaceutical composition according to the item 8, wherein the agent for treating skin diseases is an agent for promoting healing of skin wounds or an agent for treating skin ulcers.
10. A method for producing the glycosaminoglycan of the present invention, comprising the following steps of:
(a) heating a pyridine-soluble salt of glycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groups, in pyridine at a temperature not less than 100xc2x0 C. in the presence of MTSTFA for a period of time that is long enough such that substantially no sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue should be detected as determined by a chemical disaccharide analysis method in which the glycosaminoglycan is decomposed with nitrous acid, reacted with para-nitrophenylhydrazine and analyzed by high performance liquid chromatography,
(b) evaporating the pyridine from the reaction mixture obtained in the step (a), and
(c) adding water to the reaction mixture obtained in the step (b) and then placing the mixture under reduced pressure at an ordinary temperature.
Embodiments of the present invention will now be described.
In the present invention, the xe2x80x9cglycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groupsxe2x80x9d is a glycosaminoglycan having sulfate groups among the glycosaminoglycans having a heparin structure of a repetitive structure of a uronic acid residue and a glucosamine residue, and includes heparin, heparan sulfate and sulfated hyaluronic acid. The xe2x80x9cglucosamine residuexe2x80x9d also include those having an acetylated amino group and a sulfated amino group.
1. Glycosaminoglycan of the Present Invention
In accordance with one aspect of the present invention, there is provided a glycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groups, wherein substantially no sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue in the backbone structure is detected as determined by a chemical disaccharide analysis method in which the glycosaminoglycan is decomposed with nitrous acid, reacted with para-nitrophenylhydrazine (also referred to as PNP-hydrazine) and analyzed by high performance liquid chromatography (hereinafter also abbreviated to as xe2x80x9cHPLCxe2x80x9d), and the molar % of a uronic acid residue having a sulfate group at the 2-position is not less than 45% relative to the total uronic acid residues, the molar % being calculated from a disaccharide composition obtained by an enzymatic disaccharide analysis method in which the glycosaminoglycan is digested with glycosaminoglycan-degrading enzymes and analyzed by high performance liquid chromatography. The glycosaminoglycan more preferably has an effective disaccharide yield of not less than 60% as described below.
As described later with reference to Test Method 1, the chemical disaccharide analysis method mentioned above refers to a method comprising decomposing, with nitrous acid, the material to be measured, reacting the product with para-nitrophenylhydrazine and analyzing the product by HPLC.
The description that substantially no sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue is detected, usually means that, in the aforementioned chemical disaccharide analysis method, it is not possible to detect a peak for ISMS (IdoA(2S)xcex11xe2x86x924AnMan(6S)-PNP where AnMan(6S)-PNP denotes AnMan(6S)xe2x80x94CHxe2x95x90Nxe2x80x94NHxe2x80x94PNP and AnMan(6S) denotes 2,5-anhydromannose-6-O-sulfate) produced by the above chemical treatment by the ordinary HPLC. Specifically, it can be determined by using as an index, a percentage of number of all the glucosamine residues not having a 6-O-sulfate group relative to the total glucosamine residue number in the glycosaminoglycan of the present invention that is calculated from an area of the ISM (IdoA(2S)xcex11xe2x86x924AnMan-PNP where AnMan-PNP denotes AnManxe2x80x94CHxe2x95x90Nxe2x80x94NHxe2x80x94PNP and AnMan denotes 2,5-anhydromannose) peak and an area of the ISMS peak. For example, not less than 95% can be considered to signify xe2x80x9csubstantially not detected,xe2x80x9d and 100% to be the most preferred. For reduction of the anticoagulative action, it is preferable that there is substantially no glucosamine residue with the 6-O-sulfate group in the structure of the glycosaminoglycan of the invention. For convenience, hereinbelow the ratio of desulfation at the 6-position of the glucosamine residue will be referred to as the xe2x80x9c6-desulfation ratio.xe2x80x9d
The 6-desulfation ratio can also be calculated from signal intensity obtained in the nuclear magnetic resonance spectrometry as described in the examples. Results obtained in this way are substantially in agreement with the xe2x80x9c6-desulfation ratioxe2x80x9d obtained by the aforementioned chemical disaccharide analysis method.
The position and the quantity of sulfate groups bound to the constituent sugar residues in the heparin structure of the glycosaminoglycan of the present invention can be calculated from a composition (disaccharide composition) of unsaturated disaccharides detected by an enzymatic disaccharide analysis method in which the glycosaminoglycan is digested with glycosaminoglycan-degrading enzymes and analyzed by high performance liquid chromatography (enzymatic disaccharide analysis method utilizing a combination of digestion with glycosaminoglycan-degrading enzymes and HPLC).
The xe2x80x9cmolar % of a uronic acid residue having a sulfate group at the 2-position relative to total uronic acid residuesxe2x80x9d means, taking as 100% the total amount of the unsaturated disaccharides expressed by the general formula mentioned below [total (molar number) of 2-acetamido-2-deoxy-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-OSxe2x80x9d), 2-deoxy-2-sulfamino-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-NSxe2x80x9d), 2-acetamido-2-deoxy-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-6Sxe2x80x9d), 2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-USxe2x80x9d), 2-deoxy-2-sulfamino-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-di(6,N)Sxe2x80x9d), 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-di(U,N)Sxe2x80x9d), 2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-di(U,6)Sxe2x80x9d), and 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter referred to as xe2x80x9cxcex94DiHS-tri(U,6,N)Sxe2x80x9d], a ratio of the aforementioned unsaturated disaccharides each having the sulfate group at the 2-position of the uronic acid residue (total (molar number) of xcex94DiHS-US, xcex94DiHS-di(U,N)S, xcex94DiHS-di(U,6)S, and xcex94DiHS-tri(U,6,N)S) represented in terms of percentage as determined in the analysis by the enzymatic disaccharide analysis method utilizing the combination of digestion with glycosaminoglycan-degrading enzymes and HPLC. To maintain the high activity for treatment of skin diseases of the glycosaminoglycan of the present invention, this value is usually not less than 45%, preferably not less than 50%, and more preferably not less than 60%. The disaccharide analysis method utilizing the combination of enzymatic digestion and HPLC refers to the enzymatic disaccharide analysis method utilizing a combination of enzymatic digestion and HPLC described in Test Method 2 later. 
The structures denoted by the above abbreviations can also be represented as shown below: xcex94DiHS-OS: xcex94HexA1xe2x86x924GlcNAc; xcex94DiHS-NS: xcex94HexA1xe2x86x924GlcNS; xcex94DiHS-6S: xcex94HexA1xe2x86x924GlcNAc(6S); xcex94DiHS-US: xcex94HexA(2S)1xe2x86x924GlcNAc; xcex94DiHS-di(6,N)S: xcex94HexA1xe2x86x924GlcNS(6S); xcex94DiHS-di(U,N)S: xcex94HexA(2S)1xe2x86x924GlcNS; xcex94DiHS-di(U,6)S: xcex94HexA(2S)1xe2x86x924GlcNAc(6S); xcex94DiHS-tri(U,6,N)S: xcex94HexA(2S)1xe2x86x924GlcNS(6S).
In the above formulas, xcex94HexA represents unsaturated hexuronic acid, GlcNAc represents N-acetylglucosamine, GlcNS represents N-sulfated glucosamine, and binding positions of sulfate groups are shown in the parentheses.
The numerical values obtained by the enzymatic disaccharide analysis method reflect the position and number of sulfate groups of the glycosaminoglycan prior to enzymatic digestion. For more accurate reflection, the enzymatic digestion has to be more uniform and the digestibility (enzymatic digestibility (Test Method 4 described below)) as high as possible, usually not less than 60%, preferably not less than 70%, and more preferably not less than 80%.
Further, the effective disaccharide yield of the glycosaminoglycan that is calculated by means of the enzymatic disaccharide analysis method, and shows the proportion of disaccharide units that can be identified by the method within the glycosaminoglycan to be the object of the analysis. The effective disaccharide yield, which is an index of the ease of structure identification, is usually not less than 60%, preferably not less than 70%, and more preferably not less than 80%.
The xe2x80x9ceffective disaccharide yieldxe2x80x9d is a value expressed as a percentage, obtained by multiplying a ratio of the total area of the peaks of the identifiable unsaturated disaccharides (xcex94DiHS-OS, xcex94DiHS-NS, xcex94DiHS-6S, xcex94DiHS-US, xcex94DiHS-di(6,N)S, xcex94DiHS-di(U,N)S, xcex94DiHS-di(U,6)S and xcex94DiHS-tri(U,6,N)S) to the total area of the peaks of the unsaturated disaccharides detected by HPLC used in the aforementioned disaccharide analysis method, by an enzyme digestibility.
In the case of the glycosaminoglycan of the present invention, unsaturated disaccharides that contain a glucosamine residue having a sulfate group at the 6-position (the total of xcex94DiHS-6S, xcex94DiHS-di(6,N)S, xcex94DiHS-di(U,6)S and xcex94DiHS-tri(U,6,N)S) is not more than 10 mol %, preferably not more than 5 mol %, and xcex94DiHS-tri(U,6,N)S is not more than 1.5 mol %, preferably not more than 1 mol %, and more preferably it is undetectable, as analyzed by the enzymatic disaccharide analysis method (in the disaccharide composition).
The 6-desulfation ratio of the glycosaminoglycan of the present invention calculated by using the standard heparin mentioned below as a reference is normally not less than 90%, when analyzed by the enzymatic disaccharide analysis method.
Heparin has been known to show interaction with (affinity for) various cytokines (for example, fibroblast growth factor, hepatocyte growth factor, vascular endothelial cell growth factor, transforming growth factor, epidermal growth factor, midkine, interleukin 8, vitronectin, heparin-binding brain cell mitogenic factor, and heparin-binding neurite outgrowth-promoting factor and so forth), and it has been known that sulfate groups bound to the heparin structure play a major part in such interactions. Because the glycosaminoglycan of the present invention has substantially no 6-O-sulfate group of the glucosamine residue, although the anticoagulative action, the affinity for the ATIII that plays a major part in that action, and the hemorrhagic action are lost, the heparin interaction with (affinity for) the aforementioned cytokines are maintained. In the glycosaminoglycan of the invention, therefore, to maintain the affinity, the molar % of the unsaturated disaccharides having glucosamine residues with a sulfate group bound to the amino group at the 2-position (the total of xcex94DiHS-NS, xcex94DiHS-di(6,N)S, xcex94DiHS-di(U,N)S and xcex94DiHS-tri(U,6,N)S) is preferably not less than 65 mol %, more preferably not less than 75 mol %, in the composition (disaccharide composition) of unsaturated disaccharides as obtained by using the above-mentioned enzymatic disaccharide analysis method. And as described above, the molar % of the uronic acid residue having a 2-O-sulfate group relative to the total uronic acid residues is normally not less than 45%, preferably not less than 50%, most preferably not less than 60%.
Moreover, to maintain the affinity for the cytokines, when sodium 3-(trimethylsily)propionate (hereinbelow abbreviated to xe2x80x9cTSPxe2x80x9d) is used as a reference (0 ppm) in the structural analysis by 13C-nuclear magnetic resonance (NMR) spectrometry using a deuterium oxide solution (described in Example 3 below), it is preferred that substantially no peak should be observed at 66.5 to 67.5 ppm, while a peak is observed at 70.0 to 71.0 ppm, and it is further preferred that, when the signal intensities around 98.3 ppm, 100 ppm and 102 ppm are compared, both of the signal intensities around 100 ppm and 102 ppm should be higher than the signal intensity around 98.3 ppm. In one of the most preferred embodiments of the glycosaminoglycan of the present invention, no continuous peak is observed in a region of from 96.5 to 97.0 ppm in addition to the aforementioned characteristics.
The glycosaminoglycan of the present invention preferably has high activity for promoting activities of the aforementioned cytokines, inter alila, the activity of basic fibroblast growth factor (bFGF) (cell proliferation-promoting activity), and it preferably has an activity for promoting bFGF activity corresponding to not less than 80%, more preferably not less than 90% of the bFGF activity-promoting activity of a standard heparin or a commercially available heparin as determined by the method for measuring bFGF activity-promoting activity in which the bFGF activity-promoting activity is measured for cultured cells subjected to cell proliferation inhibition by using NaClO3 (see Test Method 9 in Examples, Measurement 1 for bFGF activity-promoting activity). Furthermore, the glycosaminoglycan of the present invention also preferably has an activity for promoting bFGF activity corresponding to not less than 70%, more preferably not less than 80%, most preferably not less than 90% of the bFGF activity-promoting activity of a standard heparin or a commercially available heparin as determined by the method for measuring bFGF activity-promoting activity in which the bFGF activity-promoting activity is measured for cultured cells cultured without using NaClO3 (see Test Method 9 in Examples, Measurement 2 for bFGF activity-promoting activity).
Because the highly-sulfated region (sulfated cluster) in the backbone structure of heparin strongly concerning the anticoagulative activity and the hemorrhagic activity is detected as xcex94DiHS-tri(U,6,N)S by the aforementioned disaccharide analysis method, in the glycosaminoglycan of the present invention, xcex94DiHS-tri(U,6,N)S is not more than 1.5 mol %, preferably not more than 1 mol %, most preferably it is undetectable, in the composition (disaccharide composition) of unsaturated disaccharides as determined by the aforementioned enzymatic disaccharide analysis method. Such glycosaminoglycan of the present invention has substantially lost the anticoagulative activity and the hemorrhagic activity.
The glycosaminoglycan of the present invention preferably has an average molecular weight of 3,000-30,000 more preferably 5,000-20,000, most preferably 7,000-16,000 as determined by using gel filtration, but it is not particularly limited.
As mentioned above, the glycosaminoglycan of the present invention has substantially lost the anticoagulative activity and the hemorrhagic activity. That is, when the activated partial thromboplastin time (also abbreviated as xe2x80x9cAPTTxe2x80x9d hereinafter) and the thromboplastin time (also abbreviated as xe2x80x9cTTxe2x80x9d hereinafter) are measured with addition of the glycosaminoglycan of the present invention at a final concentration of 30 xcexcg/ml in a reaction mixture in the measurement methods of APTT and TT (Test Methods 5 and 6), APTT does not exceed 50 seconds, and with addition at a final concentration of 100 xcexcg/ml, TT does not exceed 50 seconds.
Furthermore, in the measurement of antithrombin activity using bovine ATIII (for example, the method described in [Measurement method for antithrombin activity] in the Test Method mentioned below (Test Method 7) and so forth), a concentration affording 50% inhibition (IC50) is preferably not less than 50 xcexcg/ml, more preferably not less than 100 xcexcg/ml.
Because the glycosaminoglycan of the present invention has substantially lost the anticoagulative activity and the hemorrhagic activity as mentioned above, and has excellent wound healing-promoting activity and skin ulcer treatment activity as will be demonstrated in the examples mentioned below, it is useful as an active ingredient of pharmaceuticals.
While the glycosaminoglycan of the present invention can also be used in a free form, it is preferably obtained as a pharmaceutically acceptable salt. Examples of such a salt include, for example, those pharmaceutically acceptable salts selected from alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, ammonium salts, amine salts such as tributylamine salts and so forth, but alkali metal salts, in particular, sodium salts are preferred.
2. Inhibitor of the Present Invention
The inhibitor of the present invention is a fructose-1,6-bisphosphate aldolase inhibitor characterized by containing the glycosaminoglycan of the present invention as an active ingredient.
The aforementioned glycosaminoglycan of the present invention which can be used as an active ingredient of the inhibitor of the present invention exhibits high affinity for fructose-1,6-bisphosphate aldolase (abbreviated as xe2x80x9cFPAxe2x80x9d hereinafter) known as an enzyme which controls reaction rates of glycolysis enzymes, and has an activity for inhibiting the reaction of the enzyme. Therefore, the glycosaminoglycan of the present invention can inhibit the whole glycolysis pathway, and hence it can be used as an active ingredient of a glycolysis inhibitor, in particular, an FPA inhibitor.
As demonstrated in the examples mentioned below, it was found that any of (1) the glycosaminoglycan of the present invention, (2) a derivative corresponding to heparin from which only the sulfate group bound to the hydroxyl group at the 2-position of the uronic acid residue of heparin through the ester bond is removed (2ODSH), and (3) a derivative corresponding to heparin from which only the sulfate group bound to the amino group to the 2-position of the glucosamine residue of heparin through the amide bond is removed (NDSH) showed affinity for FPA and inhibits the activity of FPA. While these results indicated that heparin showed the highest activity for inhibiting FPA, it was also found that, among the substances of (1), (2) and (3), the glycosaminoglycan of the present invention showed the highest FPA inhibitory activity, the derivative of (2) showed secondly high inhibitory activity, and the derivative of (3) showed the weakest inhibitory activity. Therefore, those experimental results indicate that it is most important to have the sulfate group at the amino group at the 2-position of the glucosamine residue in the heparin structure, it is secondary important to have the sulfate group bound to the hydroxyl group at the 2-position of the uronic acid residue in the heparin structure for the FPA activity, and the sulfate group of the least involvement is the sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue through the ester bond. The present invention was accomplished based on these findings, and the inhibitor of the present invention is an FPA inhibitor containing, as an active ingredient, the glycosaminoglycan of the present invention having high FPA inhibitory activity comparable to that of heparin and reduced side effects shown by heparin such as hemorrhagic activity.
The glycosaminoglycan of the present invention used as an active ingredient of the FPA inhibitor preferably contains not less than 40 mol % of a glucosamine residue of which amino group at the 2-position is sulfated, relative to the total amount of glucosamine residues constituting the backbone structure of glycosaminoglycan. More specifically, in the aforementioned enzymatic disaccharide analysis method (disaccharide composition), the molar % of unsaturated disaccharides containing glucosamine residues having the sulfate group at the 2-position (xcex94DiHS-NS, xcex94DiHS-di(6,N)S, xcex94DiHS-di(U,N)S, xcex94DiHS-tri(U,6,N)S) is preferably not less than 40 mol %, more preferably not less than 50 mol %. Further, the glycosaminoglycan of the present invention used as an active ingredient of the FPA inhibitor preferably contains not less than 45 mol % of a uronic acid residue of which hydroxyl group at the 2-position is sulfated, relative to the amount of uronic acid residues constituting the backbone structure of the glycosaminoglycan. More specifically, in the aforementioned enzymatic disaccharide analysis method (disaccharide composition), the molar % of uronic acid residues having the sulfate group at the 2-position (xcex94DiHS-US, xcex94DiHS-di(U,6)S, xcex94DiHS-di(U,N)S, xcex94DiHS-tri(U,6,N)S) is normally not less than 45 mol %, preferably not less than 50 mol %.
The glycosaminoglycan of the present invention used as an active ingredient of the FPA inhibitor preferably has Ki values of less than 0.4 xcexcg/ml and less than 2.5 xcexcg/ml for the isozymes A4 and C4 of bovine brain fructose-1,6-bisphosphate aldolase, respectively, as determined under the conditions mentioned in Example 15 described below.
The inhibitor of the present invention can be used as agents for treatment and prevention of diseases or disorders accompanied by hypermetabolism of the glycolysis pathway. Examples of such diseases or disorders accompanied by hypermetabolism of the glycolysis pathway include, for example, infection of malaria parasite and so forth, and pharmaceutials containing the inhibitor of the present invention are considered to exhibit therapeutic or prophylactic effect for such diseases or disorders.
3. Pharmaceutical Composition of the Present Invention
The pharmaceutical composition of the present invention is characterized by containing the glycosaminoglycan of the present invention as an active ingredient.
Because the aforementioned glycosaminoglycan of the present invention is substantially free from the anticoagulative activity and the hemorrhagic activity which are considered to become problems as side effects, it can be administered to a living body as a drug.
Among the physiological activities possessed by the glycosaminoglycan of the present invention, particularly remarkable are activities for promoting tissue wound or ulcer healing, and the glycosaminoglycan of the present invention can be utilized as an agent for treatment (including prophylactic agent) of wounds and ulcers of tissues. The aforementioned tissue include epithelial tissues such as skin, cornea and mucosae including nasal cavity mucosa and oral cavity mucosa, connective tissues such as cartilage and bone and nervous tissues, as well as alimentary tract and blood vessel tissues and so forth. The wound includes disorders occurring in the aforementioned tissues (e.g., injury caused by an external force, wound of burn etc.). Among those, a preferred embodiments of the pharmaceutical composition of the present invention is, in particular, an agent for promoting healing of wounds or ulcers produced on skin (agent for treatment of skin diseases), and the most preferred embodiments of the pharmaceutical composition of the present invention are an agent for promoting healing of skin wounds and an agent for treatment of skin ulcers. The aforementioned skin ulcer include, for example, in addition to usual ulcers, intractable skin ulcers such as lower extremity ulcer and decubital ulcer (diabetic skin ulcer is also included).
Because the glycosaminoglycan of the present invention exhibits high affinity for bFGF and high activity for promoting its activity as demonstrated in the examples described below, the pharmaceutical composition of the present invention can also be utilized in embodiments intended to be applied to disorders and diseases healing of which is considered to involve bFGF, in addition to the aforementioned embodiments, and it exhibits more excellent effect compared with pharmaceuticals containing known modified heparins. Examples of such disorders and diseases include, for example, periodontal disease, restenosis, cancer, diseases involving neovascularization (proliferating retinitis, rheumatoid arthritis, psoriasis etc.), ischemic reperfusion disorder, inflammation, various circulatory organ diseases and so forth.
Moreover, as explained for the inhibitor of the present invention, because the glycosaminoglycan of the present invention has FPA inhibitory activity, it can be utilized as an agent for treatment and prevention of diseases accompanied by hypermetabolism of the glycolysis pathway.
Therefore, skin diseases can be treated and diseases accompanied by hypermetabolism of the glycolysis pathway can be treated or prevented by administering an effective amount of the glycosaminoglycan of the present invention to a subject in need of treatment of skin diseases or treatment or prevention of diseases accompanied by hypermetabolism of the glycolysis pathway.
Dosage forms and administration routes used when the pharmaceutical composition of the present invention is administered to a living body can be suitably selected depending on characteristics and severity of diseases of interest. For example, the glycosaminoglycan of the present invention can safely be administered parenterally or orally as it is, or in the form of a pharmaceutical composition containing other pharmaceutically acceptable carriers, excipients, diluents and so forth (for example, external preparations such as solutions, suspensions, ointments, plasters, lotions, pastes, liniments and patches, and injections, suppositories, tablets, capsules and so forth) to warm blooded animals (for example, human, mouse, rat, hamster, rabbit, dog, cat, horse and so forth).
When the glycosaminoglycan of the present invention is utilized as an agent for treatment of skin diseases, parenteral administration is particularly preferred, and dosage forms suitable for such administration include the aforementioned external preparations. Administration can be attained by, but not limited to, dropping, application, plastering and so forth.
Formulating amounts in the compositions and administration amounts of the glycosaminoglycan of the present invention should be individually decided depending on administration schemes of the preparations, dosage forms, specific conditions of patients, body weight of patients and so forth, and they are not particularly limited. However, the administration amount of the glycosaminoglycan of the present invention is generally, for example, about 100 xcexcg/kg to about 100 mg/kg a day. As for administration frequency of the preparations, it may be one time a day, or it may be 2-4 times or more a day.
The amount of the glycosaminoglycan of the present invention to be added to the pharmaceutical composition of the present invention varies depending on the dosage form of the composition. It may be, for example, about 10 xcexcg to 50 mg per unit of the composition, but it should be suitably controlled depending on the dosage form, specific conditions of patients and so forth.
The 50% lethal dose (LD50) of normal heparin determined by an acute toxicity test in mice (male and female) has been known to be not less than 5,000 mg/kg for oral administration, not less than 2,500 mg/kg for subcutaneous or intraperitoneal administration, or not less than 1,000 mg/kg for intravenous injection, and it is generally used as a drug (anticoagulant) at present. Therefore, its safety has already been established.
On the other hand, the glycosaminoglycan of the present invention used for the pharmaceutical composition of the present invention showed no death when it was administered to normal rats and mice with hereditary diabetes as indicated in the examples mentioned below. Further, the glycosaminoglycan of the present invention is a substance produced based on heparin, and shows extremely reduced anticoagulative activity and hemorrhagic activity as compared with heparin or has substantially lost these activities. Therefore, the pharmaceutical composition of the present invention containing the glycosaminoglycan of the present invention can be said to be highly safe for warm blooded animals.
4. Production Method of the Present Invention
The production method of the present invention is a method for producing the aforementioned glycosaminoglycan of the present invention, and includes the following steps:
(a) heating a pyridine-soluble salt of glycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groups, in pyridine at a temperature not less than 100xc2x0 C. in the presence of MTSTFA for a period of time that is long enough such that substantially no sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue should be detected as determined by the enzymatic disaccharide analysis method,
(b) evaporating the pyridine from the reaction mixture obtained in the step (a), and
(c) adding water to the reaction mixture obtained in the step (b) and then placing the mixture under reduced pressure at an ordinary temperature.
An example of the production method of the present invention will be explained below.
(a) Step of Heating a Pyridine-soluble Salt of Heparin in Pyridine at a Temperature not Less than 100xc2x0 C. in the Presence of MTSTFA
The xe2x80x9cpyridine-soluble salt of glycosaminoglycan having a backbone structure comprising a repetitive disaccharide bearing a uronic acid residue and a glucosamine residue, and having sulfate groupsxe2x80x9d used in the production method of the present invention is preferably a pyridinium salt of heparin, while it is not particularly limited so long as it is a pyridine-soluble salt of heparin. Such a pyridinium salt of heparin can be obtained by, for example, passing sodium salt of heparin thorough a cation exchange column (for example, a column packed with Amberlite IR-120B (H+ form) resin (produced by ORGANO CORP.)) which is equilibrated with distilled water to be converted into a free form, and adding excessive pyridine to the resulted acidic fraction to adjust pH thereof to 5 to 7, preferably 5.5 to 6.5, and lyophilizing the fraction. A commercially available heparin pyridinium salt may also be used.
The aforementioned pyridine-soluble salt of heparin is reacted in pyridine in the presence of MTSTFA. The MTSTFA used for the reaction is added in an amount of normally 6- to 12-fold volume (w/w), preferably 8- to 11-fold volume (w/w) of the pyridine-soluble salt of heparin. The amount of pyridine used for the reaction is preferably 5- to 150-fold volume (v/w), preferably 10- to 120-fold volume (v/w), most preferably 15- to 110-fold volume (v/w) of the pyridine-soluble salt of heparin, but it is not limited to these amounts.
Temperature for the aforementioned reaction is preferably 100-115xc2x0 C., most preferably 108-112xc2x0 C. The period of time that is long enough such that substantially no sulfate group bound to the hydroxyl group at the 6-position of the glucosamine residue should be detected in the chemical disaccharide analysis method is, when a temperature within the aforementioned preferred temperature range is maintained, preferably 90 to 150 minutes, most preferably 100 to 130 minutes. Moreover, by maintaining the aforementioned temperature for 10-20 minutes, 25-35 minutes, or 50-70 minutes, for example, it is also possible to prepare a glycosaminoglycan having about 50%, about 70%, or about 90% of 6-desulfation ratio, respectively. That is, in the production method of the present invention, it is possible to precisely control the 6-desulfation ratio by controlling the reaction time as described above.
After the completion of the aforementioned reaction, the reaction is preferably stopped by cooling the reaction mixture. Such cooling can be attained by, for example, leaving a vessel containing the reaction mixture at room temperature, or cooling it with flowing water or ice. While the method for cooling is not particularly limited, it is preferably attained by ice cooling.
(b) Step of Evaporating Pyridine
The pyridine is evaporated from the cooled reaction mixture. While the pyridine can be evaporated by any known method for evaporating organic solvents, it is preferably performed by using an evaporator at 25 to 37xc2x0 C. under reduced pressure, because of ease of its operation. The reaction mixture is concentrated by the evaporation. As for the degree of concentration of the reaction mixture, it is preferably 7- to 25-fold concentration, most preferably 8- to 20-fold concentration of the reaction mixture.
The term xe2x80x9creduced pressurexe2x80x9d usually means a pressure of 10xe2x88x922 to 10xe2x88x924 Torr.
(c) Step of Adding Water and Placing under Reduced Pressure at Ordinary Temperature
To the reaction mixture concentrated through the evaporation of pyridine, water is added in order to decompose MTSTFA bound to hydroxyl groups and free MTSTFA. The amount of water to be added is preferably 1.5- to 3-fold amount, most preferably 1.8- to 2.5-fold amount with respect to the concentrated reaction mixture. When water is added to the concentrated reaction mixture as described above, white turbidity is produced in the reaction mixture. To eliminate the white turbidity, the concentrated reaction mixture to which water is added is placed under reduced pressure at an ordinary temperature. The reduced pressure at an ordinary temperature can be realized by using an evaporator, and the reduced pressure is preferably maintained at 25 to 37xc2x0 C. until the white turbidity disappears. The reduced pressure is normally maintained for 5 to 10 minutes. The reduced pressure is preferably a pressure of 10xe2x88x922 to 10xe2x88x924 Torr like the aforementioned reduced pressure, and it is preferably a pressure under which the concentrated reaction mixture is boiled because of the reduced pressure.
(d) Other Steps
After the treatment of the aforementioned step (c), decomposition products of MTSTFA and the organic solvent are preferably removed from the reaction mixture. To this end, known methods can be used, which include methods utilizing dialysis, ethanol precipitation, cation exchange column and so forth.
When dialysis is used, only flowing water or a combination of flowing water and distilled water can be used for the outer liquid of the dialysis. When dialyzed against flowing water, the dialysis is normally performed for at least 24 hours, preferably at least 40 hours, most preferably 48 hours. When dialysis is performed by using a combination of flowing water and distilled water, the reaction mixture can be dialyzed against flowing water, and then dialyzed against distilled water. After the dialysis is performed against flowing water as described above, the dialysis against distilled water is normally performed for at least 1 hour, preferably 1.5 to 2.5 hours.
From the reaction mixture from which the decomposition products of MTSTFA and the organic solvent are removed, the glycosaminoglycan of the present invention can be obtained in the form of a salt thereof by a usual method for precipitating glycosaminoglycans.
As the method for obtaining the glycosaminoglycan of the present invention as a salt, there can be mentioned, for example, a method comprising fractionating the inner solution obtained from the dialysis by using a cation exchange column (for example, a column packed with Amberlite IR-120B (H+ form) resin (produced by ORGANO CORP.)) equilibrated with distilled water to collect an acidic fraction, adjusting pH of the acidic fraction to 8 to 10, preferably 8.5 to 9.5 by adding an aqueous alkaline solution (for example, aqueous alkali metal hydroxide or alkaline earth metal hydroxide such as aqueous sodium hydroxide, aqueous potassium hydroxide, aqueous magnesium hydroxide, and aqueous calcium hydroxide preferably at a concentration of 0.1-2 N), dialyzing the fraction against flowing water for normally at least 15 hours, preferably about 18 hours, then against distilled water for normally 1.5 to 2.5 hours, and lyophilizing the inner solution obtained from the dialysis. By this method, a lyophilized product of the glycosaminoglycan of the present invention can be obtained.
A modified version of the glycosaminoglycan of the present invention with different degrees of sulfation at the 2-position of the glucosamine residue and the 2-position of the uronic acid residue can be prepared by changing such sulfation degrees of the glycosaminoglycan of the present invention as required by appropriately using a method comprising sulfating the amino group at the 2-position of the glucosamine residue or the hydroxyl group at the 2-position of the uronic acid residue constituting the backbone structure, and a method comprising releasing the sulfate group at the 2-position of the uronic acid residue constituting the backbone structure in combination.
As the method for sulfating the amino group at the 2-position of the glucosamine residue, for example, the method of Nagasawa et al. (Carbohydr. Res., (1989) 193, 165-172) with modification can be mentioned. That is, the glycosaminoglycan of the present invention or a salt thereof is dissolved in an alkaline solution at about pH 9 to 10 (for example, sodium carbonate solution, sodium hydroxide solution, potassium hydroxide solution etc.), and solid trimethylammonium sulfonate or triethylammonium sulfonate is additionally added at 50 to 55xc2x0 C. over 6 to 24 hours.
Further, as the method for sulfating the hydroxyl group at the 2-position of the uronic acid residue, for example, there can also be mentioned the method of Nagasawa et al. (Carbohydr. Res. (1989) 193, 165-172), which is the same method as mentioned above, with modification. That is, the glycosaminoglycan of the present invention is made into a tributylammonium salt by salt exchange in a conventional manner, and the obtained tributylammonium salt of the glycosaminoglycan of the present invention is fully dissolved in N,N-dimethylformamide, and allowed to react with 5 to 20 molar equivalents/(mole of free hydroxyl groups) of sulfated pyridine at xe2x88x9210 to 0xc2x0 C. for 1 hour. However, in this method, along with the sulfation of the hydroxyl group at the 2-position of the uronic acid residue, and the sulfate group at the 6-position of the glucosamine residue are also sulfated. Therefore, when this method is used, it is preferable to subject the product to the method for producing the glycosaminoglycan of the present invention again to release the sulfate group at the 6-position of the glucosamine residue.
As the method for releasing the sulfate group at the 2-position of the uronic acid residue, there can be mentioned, for example, a partially modified version of the method of Jaseja et al. (Can. J. Chem. (1989) 67, 1449-1456). That is, a sodium salt of the glycosaminoglycan of the present invention is dissolved in a NaOH solution, and lyophilized immediately. The obtained lyophilized powder is dissolved in distilled water, and adjusted to pH 6 to 8, preferably pH 6.5 to 7.5, by addition of acetic acid. Then, this solution is subjected to dialysis and lyophilized.
A preparation of the glycosaminoglycan of the present invention preferably contains less contaminated endotoxins. In particular, the amount of such contaminated endotoxins (endotoxin activity) contained in 1 mg of a preparation of the glycosaminoglycan of the present invention is preferably, but not limited to, not more than 0.2 USP endotoxin unit (EU), more preferably not more than 0.1 EU, most preferably less than 0.05 EU.
Further, a preparation of the glycosaminoglycan of the present invention preferably has a content of residual pyridine of not more than 200 ppm, more preferably not more than 150 ppm, most preferably not more than 100 ppm, in order to secure safety when the preparation of the glycosaminoglycan of the present invention is used as a raw material of pharmaceutials. According to the Japanese Pharmacopoeia, the residual pyridine content in pharmaceutical preparations is regulated to be not more than 200 ppm.